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Journal of Herbal Medicine and Toxicology 4 (2) 167-175 (2010)
ISSN : 0973-4643 Original Article
ENHANCEMENT OF ANTIMICROBIAL POTENTIAL OF
PHYLLANTHUS NIRURI BY FERMENTATION
Vaishnavi Venugopalan, Dinesh M.S.* and Geetha K.S.
Department of Biotechnology, PES Institute of Technology, 100 feet Ring Road, BSK III Stage, Bangalore
*Corresponding author - E-mail: dineshms@pes.edu
Received - 5th April, 2010; Revised - 4th May, 2010; Accepted - 28th May, 2010
ABSTRACT: The use of plants products for medicinal value is common in Indian
system of medicine. We can enhance the medicinal property of plants throughbioprocesses. In the present study, bioprocess such as fermentation was used to enhancethe antimicrobial potential of the crude herbal extract of Phyllanthus niruri.
Fermentation was carried out using the standard/commercial isolates of Lactobacillusacidophilus and by the isolates of the same bacterium from the herb surface separately.The fermented product obtained from both these procedures were compared for their
antimicrobial property against common human pathogens like Escherichia coli,Staphylococcus aureus, Salmonella typhi, Psuedomonas aeruginosa, Bacillus cereus,Bacillus subtilus and Klebsiella species. The variation of the antimicrobial property of
the fermented extracts along the fermentation time period was also studied. The resultsindicated that the antimicrobial potential of the fermented herb is more than that ofthe crude herbal extract. The antimicrobial property of the fermented herb increasesby about 80% -170% when compared to the crude herbal extract. Also, the fermented
product obtained using Lactobacillus isolates from the herbal surface is more potentagainst tested human pathogens when compared to the product obtained usingcommercial L.acidophilus isolates. The potency improves by 49% if Lactobacillus
species from the herbal surface are used for fermentation. The antimicrobialcapabilities of the fermented product increases along the fermentation period by about65%-95% irrespective of the source of lactic acid bacteria used. Another salient feature
of the study is that E.coli is the most sensitive while Klebsiella species is the leastsensitive to both the crude as well as the fermented extracts.
Key Words: Phyllanthus niruri, Lactobacillus isolates, Fermentation, Antimicrobial
activity
INTRODUCTION
Plants have always been a common source of
medicaments, either in the form of traditional
preparations or as pure active principles. Medicinal
plants are considerably useful and economically
essential. They contain active constituents that are
used in the treatment of many human diseases [1].
Many plant extracts have been developed and
proposed for use as antimicrobial substances. Plants
used in traditional medicine contain a vast array of
substances that can be used to treat chronic and
infectious diseases [2]. Plants have an almost limitless
ability to synthesize aromatic substances, most of
which are phenols or their oxygen-substituted
derivatives. Most are secondary metabolites, of which
at least 12,000 have been isolated, a number estimated
to be less than 10% of the total [3]. In many cases,
these substances serve as plant defense mechanisms
against predation by microorganisms, insects, and
herbivores. Some, such as terpenoids, give plants their
odors; others (quinones and tannins) are responsible
for plant pigment. Many compounds are also
responsible for plant flavor while some of the herbs
and spices used by humans to season food yield useful
medicinal compounds [4]. Phyllanthus niruri is a
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168
herb belonging to Euphorbiaceae family. It is known
for a wide variety of phytochemicals and
pharmacological properties. It is an ingredient of
almost 175 ayurvedic formulations. Fruits are
commonly used in the treatment of hemorrhages,
diarrheas, dysentery, jaundice, cough and anaemia. It
is also used for the preparation of various health care
and personal products like chavanprash, hair oil, dye,
face cream, tooth powder etc [5]. The active
phytochemicals, flavonoids, alkaloids, terpenoids,
lignans, polyphenols, tannins, coumarins and saponins,
have been identified from various parts of P. niruri.
Extracts of this herb have been proven to have
therapeutic effects in many clinical studies [6].
Fermentation of herbs produces beverages which are
considered to be healthy by people who consume it.
They contain high nutritional content and bioactive
compounds formed from the raw material and the
fermentation reactions [7]. Lactic acid fermentations
is regarded the most popular used for fermenting of
herbs. Lactic acid bacteria (LAB) produces organic
acids, diacetyl, hydrogen peroxide and bacteriocins
that improve the shelf life of the fermented product
by controlling spoilage organisms and potential
pathogens. LAB is also known to possess
antimicrobial properties thereby improving human
health [8, 9]. In the present study, a crude extract of
P.niruri was tested against common human pathogens
using a standard antibiotic as positive control and
sterilized distilled water as a negative control. Further
to this, the herb was separately fermented using
Lactobacillus species isolated from two different
sources – standard or commercially available
Lactobacillus acidophilus and the Lactobacillus
sp. isolated from the herbal surface. The antimicrobial
activities of the fermented products obtained from
both these bacterial strains were then compared and
evaluated. The activities along the fermentation period
(25 days) were also studied comparatively.
MATERIAL AND METHODS
Plant material: The fresh herbs were collected from
the aromatic crop section, Division of Horticulture,
UAS (University of Agriculture Sciences), GKVK,
Bangalore, India, for the present study. Fresh herbs
that are not infected by pests and disease damage
were harvested. The parts of the herbs like leaves
and fruits were detached and washed thoroughly with
clean water.
Preparation of the crude extract: 20g of clean and
fresh herbs were crushed using a pestle and mortar
and added to 100 ml sterile water to obtain an aqueous
extract of the herb (200mg/ml). The extraction was
done at room temperature (24 °C). Muslin cloth was
then used to filter the plant residues and the filtrate
thus obtained was further purified by filtration through
Whatman No 1 filter paper [10, 11].This crude extract
was tested against common human pathogens for
antimicrobial properties.
Culturing of Lactobacillus isolates for fermentation
of the herb: Standard or commercial Lactobacillus
acidophilus isolates were obtained as pure cultures
from the Department of Microbiology, G.K.V.K,
Bangalore, and stored in semi solid medium at 4° C
for further utilization in the study. Before use, there
were thawed and maintained in Mann Rogosa
Sharpe’s media [12]. In order to isolate Lactobacillus
species from the herbal surfaces, clean and washed
leaves of the herb were placed on Mann Rogosa
Sharpe’s agar (15 ml) in petri plates under sterile
procedures [13]. MRS agar is used for isolation of
lactic acid bacteria species since the medium restricts
growth of other species [12]. The plates were
incubated at 37°C for 2 days. On observing the petri
plates, uniform colonies of bacteria were seen to be
grown. These colonies were similar in morphology
and other physical characteristics as that of standard
Lactobacillus acidophilus colonies maintained. The
colonies isolated from the herbal surfaces were
identified and characterized using standard methods
such as gram staining, test for catalase, gelatin
hydrolyses activities and ability to assimilate a range
of carbon sources [13, 14]. Hence, the standard and
the herbal isolates of Lactobacillus species were
separately used to ferment the herb extract of
Phyllanthus niruri to know their efficacies. The
characteristics of the two lactic acid bacterial isolates
are outlined in table 1. The isolates used in the study
are shown in figure 1.
Lactic acid fermentation of the herb [15-17]:
The following sequential steps were followed.
(i) Preparation of P.niruri based fermentation media:
The pest and disease free leaves of the herb were
harvested after eliminating its surface microbial load
using running water. 20% of the medicinal herb was
considered for fermentation. 20g of leaves were
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Venugopalan et al.
Table 1: Characteristics of the Lactic acid bacteria (LAB) isolates collected from two different
sources
Characters tics studies Source of LAB Isolates
Standard / Commercially
available LAB
LAB isolated from herbal
surface of Phyllanthus niruri
Cell Shape and Arrangement Rods Rods occurring in short chain
Gram Reaction Positive Positive
Catalase Reaction Negative Negative
Gelatin Hydrolysis Positive Positive
Growth on MRS broth after 24 hrs of incubation (OD at 600nm) 1.25 1.23
Growth on MRS broth after 48 hrs of incubation (OD at 600nm) 1.71 1.72
Assimilation of Glucose Good growth Good growth
Assimilation of Sucrose Medium growth Medium growth
Assimilation of Galactose Medium growth Medium growth
Assimilation of Lactose Medium growth Medium growth
Organism Lactobacillus
acidophilus
Lactobacillus Sp.
The above table shows the different characteristics studied for the two sources of Lactic acid bacteria (LAB) isolated in the
present research work – one commercially available and the other from the surfaces of the herb Phyllanthus niruri.
Table 2: Antimicrobial Activities of crude herbal extract of P.niruri and fermented products
against common human pathogens
Bacterial
Pathogens
Zone of
Inhibition
(mm) for
NC
Zone of
Inhibition
(mm) for
CEx
Zone of Inhibition (nm) at different fermentation times Zone of
Inhibition
(mm) for
PC
Day 5 Day 10 Day 15 Day 20 Day 25
A B A B A B A B A B
E. Coli 0.0 6.0 6.5 8.0 8.5 9.5 9.0 11.5 10.5 14.0 10.5 14.0 2.5
S. aureus 0.0 5.5 6.0 7.5 7.5 8.5 8.0 9.5 9.0 12.0 9.1 12.0 20.5
S. thphi 0.0 4.0 4.0 4.5 5.0 5.5 6.0 7.0 6.5 9.0 6.5 9.2 2.5
P.aeruginosa 0.0 2.0 2.5 3.0 3.5 4.0 3.5 6.5 4.0 8.5 4.1 8.5 1.5
B.cereus 0.0 1.5 2.0 2.5 2.0 3.5 3.0 5.0 3.5 6.0 3.5 6.1 14.0
B.subtilus 0.0 1.5 1.5 2.0 2.0 2.5 2.5 3.5 3.0 4.5 3.0 4.5 15.1
Klebsiella sp. 0.0 0.5 1.0 1.5 1.5 2.0 2.0 2.5 2.5 3.5 2.6 3.5 6.1
NC: Negative control (distilled water); CEx: Crude Extract of the herb Phyllanthus niruri; PC: Positive control
(Ampicillin taken 10 µg/disc); A: Fermented products obtained using Standard/commercially available LAB
isolates ; B: Fermented products obtained using LAB isolated from the herbal surfaces of Phyllanthus niruri. The
above table shows the antimicrobial activities of the controls and the samples against common human pathogens.
Fermented samples are tested every 5 days for a period of 25 days of the fermentation period. The tests were
performed in triplicates and the average value has been entered in the above table.
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Venugopalan et al.
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crushed with pestle and mortar and added to 100ml
of sterile distilled water. The extract was filtered using
muslin cloth and Whatman No 1 filter paper to remove
the plant debris present. 400 ml of this herbal extract
was made and stored in conical flasks (100 ml each),
to be used for preparing starter cultures and for lactic
acid fermentations. The Lactobacillus species
(standard isolates and herbal surface isolates)
collected and maintained previously were used
separately to ferment the herbal extract prepared.
Before subjecting the herbal extract to fermentation,
each of the Lactobacillus isolates were multiplied
and made to adapt to herbal environment by preparing
respective starter cultures. (ii) Preparation of starter
culture of Lactobacilli strains: To prepare a starter
culture, a test tube with 5ml of sterilized MRS broth
was added with one loop inoculums of the lactic acid
bacterial culture intended to be multiplied and kept
overnight at 25 °C for growth. The culture grown in
the test tube was then added to 100ml of herbal extract
in a conical flask. The flask was kept at 37 °C in an
incubator with shaking facility of 100rpm for 24 hrs.
Dilution was done using MRS broth to obtain a size
of 106 cfu/ml. Adjusted cultures was tested for right
size by plate count method on MRS agar. 5ml (v/v)
of the adjusted starter culture was used for further
study. Respective starter cultures were used for
respective fermentations keeping the number of lactic
acid bacterial cells added to herbal extract constant.
(iii) Setting up the fermentation process: 100 ml of
the herbal extract in a conical flask was fermented
using standard Lactobacillus acidophilus starter
culture and 100 ml of herbal extract in another conical
flask was fermented using starter culture of
Lactobacillus isolates from the herbal surface. About
2g of dextrose was added to enhance the fermentation
process. Fermentation process was carried out at room
temperature using a shaker incubator (100rpm). The
fermented products from each of these were
compared for antimicrobial activity at every 5 days
interval for a period of 25 days. A flow chart of the
fermentation procedure is shown in fig 1.
Collection and culturing of human pathogens: The
microorganisms to be tested against the crude herbal
extract and the fermented products of P.niruri were
obtained from the Department of Microbiology,
G.K.V.K., Bangalore, as pure cultures maintained in
nutrient agar medium at 4°C for timely use. The
human pathogens collected were Escherichia coli,
Staphylococcus aureus, Salmonella typhi,
Psuedomonas aeruginosa, Bacillus cereus,
Bacillus subtilis and Klebsiella species. Suspension
cultures of the pathogens were made prior to testing
the antimicrobial potential of the fermented products.
Nutrient broth was used for the preparation of bacterial
culture suspension [18]. Using a sterile loop, 3-5
colonies of the bacterial cells were transferred from
the stock culture plate into a 10ml of nutrient broth
taken in test. The test-tube was incubated at 37°C
for 24 hours. Using sterile nutrient broth, a population
of 106 cfu/ml was obtained for each pathogen.
Appropriate dilution was determined after spread
plating the adjusted culture on Plate Count Agar
(PCA). 100 µl of 24 hr incubated test organism was
used for further studies [19]. All the growth media
used in the experiment was purchased from Himedia
Laboratories Pvt. Ltd., Mumbai.
Screening the crude herbal extract and fermented
products for antimicrobial activity: To test the
antimicrobial potential of fermented products, modified
agar diffusion method (filter paper disc method) was
employed [20, 21]. On solid nutrient agar medium (15
ml taken in petri plate), the pathogenic culture
suspension was swabbed uniformly. A sterile filter
paper impregnated with 0.1ml of the sample to be
tested for antimicrobial activity was placed on the
agar surface and kept for incubation at 37°C for 24
hrs. After the stipulated time, presence of a clear zone
of inhibition around the filter paper disc indicated the
sensitivity of the pathogen to the sample tested [22,
23]. Sterile distilled water was taken as negative
control while ampicillin (10µg/ml) was taken as the
positive control. The zone of inhibition was calculated
by subtracting the diameter of the zone formed due
to application of the sample on the filter paper disc
from the diameter of filter paper disc used for testing
sensitivity of the pathogens to the sample [21]. The
experiment was done in triplicates and the zones of
inhibition seen were reported in mm.
RESULTS AND DISCUSSION
(i) Isolation and collection of Lactobacillus species:
The characteristics of Lactobacillus species collected
from standard sources and the ones isolated from
herbal surfaces were studied by growth on MRS agar.
These include features like colony morphology, gram
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Venugopalan et al.
staining, catalase activity, gelatin hydrolysis activity
and capability to assimilate various sugars. The results
of the same are outlined in table 1. From the table it
can be seen that species isolated from the herbal
surfaces bears same features as that of the
commercially available L.acidophilus species. Hence
it was reported that the species isolated from the herb
on MRS agar is Lactobacillus species. Literature
shows that studies of isolating lactic acid bacteria from
different herbs gave similar results as the present
study. J. Orvin Mundt and James L. Hammer isolated
Lactobacillus casei, L.leichmanii, L.platarum from
chickpeas and cowpeas in 1968 [24]. Cereals and
millets contain lactic acid bacteria species which were
successfully isolated on MRS media [25, 26].
(ii) Screening and evaluation of antimicrobial activity
of crude extract P.niruri: The antimicrobial assay
done by filter paper disc procedure showed that the
crude herbal extract of Phyllanthus niruri exhibits
antibacterial activity against all the tested pathogens.
During the assay, sterile distilled water was used as
negative control while ampicillin was used as positive
control. The zone of inhibition in mm for the crude
extract is shown in table 2. From the table it can be
seen that the zones of inhibition formed by the crude
extract and standard antibiotic (ampicillin) are
comparable. Results showed that out of the 7
microorganisms tested, E.coli, S.typhi and
P.aeruginosa were most sensitive to the crude extract
when compared to the standard antibiotic. The herb
Phyllanthus niruri is a rich source of phytochemicals,
including many which have been found only in the
Phyllanthus genus [27]. Many of the constituents are
attributed to biologically active lignans, glycosides,
flavonoids, alkaloids, ellagitannins, and
phenylpropanoids found in the leaf, stem, and root of
the plant. Common lipids, sterols, and flavonols also
occur in the plant. The main plant chemicals in it
include alkaloids, astragalin, brevifolin, carboxylic
acids, corilagin, cymene, ellagic acid, ellagitannins,
gallocatechins, geraniin, hypophyllanthin, lignans,
lintetralins, lupeols, methyl salicylate, niranthin,
nirtetralin, niruretin, nirurin, nirurine, niruriside,
norsecurinines, phyllanthin, phyllanthine, phyllanthenol,
phyllochrysine, phyltetralin, repandusinic acids,
quercetin, quercetol, quercitrin, rutin, saponins,
triacontanal, and tricontanol [28].
(iii) Enhancement of antimicrobial activity: During
fermentation of P.niruri, the antimicrobial potency
was seen to be enhanced when the herbal extract
was fermented using lactic acid bacteria. This was
evident through comparing the zones of inhibition (mm)
formed against the tested pathogens by crude extract
and the fermented products (Table 2). Fermentation
was carried out separately by using standard
L.acidophilus isolates and Lactobacillus species
isolated from the herbal surfaces. Fermented products
of both the fermentation procedures gave
antimicrobial potential better than that of the crude
unfermented extract. In comparison to the crude
extract, fermentation of the herb increased the
antimicrobial activity by 80% when commercial
L.acidophilus were used and by 170% when herbal
isolates of Lactobacillus species were used. Many
phytochemicals important for antimicrobial activity in
a herb are less freely available and mainly exist bound
to the cell surface [30, 31]. Fermentation process
causes release of microbial enzymes which in turn
produce more freely available forms of plant
chemicals like flavonoids, alkaloids and
phenylpropanoids [32]. The increase in the levels of
free (non-bound) plant chemicals may be responsible
for the improvement in antimicrobial activities. Also,
the fermented products formed by the Lactobacillus
species from the herb surfaces gave better zones of
inhibition than the products formed by standard
L.acidophilus isolates. The antimicrobial property
was enhanced by about 49% when herbal isolates of
lactic acid bacteria was used for fermentation. This
could be because the Lactobacillus species isolated
from Phyllanthus niruri are native to the herb and
hence more adapted to the herb than the commercially
available L.acidophilus. Due to this, the potential to
ferment the herbal extract is better by former than
the latter thereby releasing more active fermentation
products that are better potent against the human
pathogens. The fermented products (irrespective of
the source of lactic acid bacteria) have a potential to
be commercialized since they showed better activities
against E.coli, S.typhi and P.aeruginosa than the
standard antibiotic that was used as positive control.
A very important result through the present study is
the increase in antimicrobial activity along the
fermentation period. Assaying was done every 5 days
to a period of 25 days, during which increase in zones
of inhibition can be clearly seen from table 2. This is
regardless of the source of lactic acid bacteria used.
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174
As the fermentation time progressed, antimicrobial
activity was increased by about 65% when
commercial L.acidophilus were used and by 95%
when herbal isolates of Lactobacillus species were
used. The activity, estimated in terms of the zone of
inhibition, reached a peak on the 20th day of
fermentation and remained steady thereafter. Since
all the available raw materials get converted to
fermented products by the 20th day of fermentation
process, the zones of inhibition formed by the samples
from then on remains constant [31]. A graphical
representation has been shown in figures 3, 4 and 5.
Among the organisms tested, the sensitivity of the
pathogens varies as follows:
E.coli>S.aureus>S.typhi>P.aeruginosa>B.cereus>B.subtilis>Klebsiella
species. The outer membrane of bacteria and its
structure are responsible for differences of sensitivity
that may occur between different bacteria [33, 34].
CONCLUSION
The results of the present study show that
fermentation of the herb Phyllanthus niruri evidently
enhanced the antimicrobial properties of the herb.
Also, using the Lactobacillus species growing on the
herbal surface if used for the fermentation process
gives better results for antimicrobial property. The
fermented P.niruri obtained using isolates of
Lactobacilli from the leaf surface have high potential
of being developed as a neutraceutical to curb
infectious diseases caused by the pathogens used in
the study.
ACKNOWLEDGMENT
The authors thank the Principal, Dr. K.N.
Balasubramanium, and the Management of PES
Institute of Technology, Bangalore India, supporting
the study through PESIT Internal project. We also
thank Dr. V. Krishnamurthy, Head of the Department,
Biotechnology, for providing useful suggestions to
improve our work. Lastly we thank the Department
of Microbiology and Horticulture, G.K.V.K., Bangalore
for providing us the materials required for the study.
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